Drug Conjugate: N4

Synthesis of chemoreversible prodrugs of ara-C with variable time-release profiles. Biological evaluation of their apoptotic activity. Peter Wipf , We...
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Bioconjugate Chem. 1994, 5, 162-166

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TECHNICAL NOTES Synthesis of a Lipid/Peptide/Drug Conjugate: W-(Acylpeptidy1)-Ara-C F. M. Menger,* Y. Guo, and A. S. Lee Department of Chemistry, Emory University, Atlanta, Georgia 30322. Received September 8, 1993@

A lipidlpeptideldrug conjugate, N4-(acylpeptidyl)-ara-C,is synthesized under mild conditions to give an ara-C prodrug protected against cytidine deaminase-catalyzed deactivation. The compounds can be bound, via their hydrocarbon tails, to phospholipid membranes and examined for chymotrypsininduced drug release. A tripeptide is too short to permit efficient enzymelmembrane contact and subsequent freeing of the drug. Faster rates can, presumably, be achieved by "fine tuning" the length and polarity of the peptide spacer attached to the drug.

INTRODUCTION 1-(P-D-Arabinofuranosy1)cytosine (ara-C1) is one of the most important drugs for the treatment of human acute myeloblastic and lymphoblastic leukemia (I,2). It is also

n=12,16

rn = 0 , 1, 2

"[email protected] OH

OH

effective in combination-chemotherapy against solid tumors (3). Clinical activity of ara-C is, however, adversely affected by a cytidine deaminase-catalyzed "2-to-OH replacement that reduces the drug's half-life in the plasma (4).

Derivatives of ara-C with long-chain N4-acylgroups have been reported to have greater chemotherapeuticactivities than does ara-C itself ( 5 ) . These derivatives are resistant to cytidine deaminase. In addition, they are more efficiently incorporated into cells, and maintained there for a longer time period, than the parent drug (6). We now describe the synthesis of N4-(acylpeptidyl)derivatives of ara-C, a new class of ara-C prodrug having two key features: (a)The N4aminogroup is protected via acylation by an enzyme-removable peptide. (b) At the distal end there is attached a long hydrocarbon chain to impart lipid solubility. Two primary considerations motivated the design of these substances: (a) We wished to bind the compounds to an i n vitro phospholipid bilayer (by means of their hydrocarbon tails) and to examine how the peptide structure affected enzyme-induced drug release at the membrane surface. Peptides are useful "spacers" in that

* Author to whom correspondence should be addressed. @

Abstract published in Advance ACS Abstracts, January 15,

1994.

'Abbreviationsused: ara-C, 1-(P-D-arabinofuranosy1)cytosine; TBDMSCl, tert-butyldimethylsilyl chloride; EEDQ, 2-ethoxyl-(ethoxycarbonyl)-l,2-dihydroquinoline; TBAF, tetrabutylammonium fluoride; DMAP, 4-(dimethy1amino)pyridine;THF, tetrahydrofuran; 3-NBA, 3-nitrobenzylalcohol. 1043-1802/94/2905-0 162$04.50/0

they can be readily modified in length and polarity. (b) We also wished to determine how i n vivo activity depended upon the tail length and peptide sequence. These studies are in progress. For the present purposes, the focus will be mainly on the synthetic details involved in assembling lipid, peptide, nucleic acid base, and sugar (the four major building blocks of life) into a single unit. In 1991, Wipf et al. (7) also synthesized N4-peptidyl derivatives of ara-C, but their work differs from ours in two important ways. First, their compounds lack a lipid moiety. Second, their method for attaching the peptide to the drug involved reagents such as LiOH and BuLi that are too harsh for our more sensitive peptides. RESULTS AND DISCUSSION Acylpeptide was formed in 64-82 5% yield by treating a fatty acid with ethyl chloroformate and triethylamine for 20 min at 0 "C in T H F (8). A 1:l aqueous solution of peptide and NaOH was then added and the mixture stirred at room temperature for 19 h. Workup consisted of quenching with 10% aqueous HC1 to pH 2, extraction into CH2C12, removal of solvent, and trituration with ether or CH&N to remove unreacted reagents. Products were characterized by NMR and elemental analysis. The remainder of the synthesis was carried out according to the three steps in Scheme 1:(a) ara-C was protected with TBDMSCl using the Wipf procedure (7), and (b) trisilylated nucleotide 1 thus obtained was reacted with an acylpeptide (1 equiv), EEDQ (1 equiv), and pyridine (6 equiv) in chloroform at 37 "C for 48 h (9). Removal of the solvent and pyridine under vacuum gave an oily product ' which was flash chromatographed on a silica column (20% CH30H in CHC4 eluant). The resulting residue was treated, without further purification, with 1 M TBAF in T H F (6 equiv) at room temperature for 2 h according to 0 1994 American Chemical Society

Technical Notes

Bioconjugate Chem., Vol. 5, No. 2, 1994

Scheme 1.

9

I

~TBDMS

OH

1

'

V

O

H

i)H 2

Key: (i) TBDMSC1, imidazole, DMAP, rt, 72 h; (ii) acylpeptide, EEDQ, py, 37 "C, 48 h; (iii) TBAF, rt, 2 h. a

a procedure of Corey and Venkateswarlu (IO). Flash chromatographyon silica (20% CH30H in CHC13) afforded the final products 2 that were characterized by 'H and 13C NMR, elemental analysis, and MS (FAB, 3-NBA matrix). Although the overall yields in Scheme 1were modest at best (21-47 % 1, attempts to conjugate the weakly nucleophilic ara-C amino group to peptides by other methods were unsuccessful altogether. All ara-C derivatives ( n = 12, 16; m = 0-2) were too water-insoluble in the absence of a solubilizing entity (micelle, vesicle, etc.) to allow a study of their a-chymotrypsin-catalyzed hydrolyses. In order to obtain "baseline" parameters, we used a short-chain analog ( n = 5, m = 0) that was sufficiently water-soluble by itself. This substrate has a kat = 5.3 s-1 and K,,, = 1.4 X 10-3 M (pH = 7.0, 25.0 "C). When the long-chained lipidfpeptidel ara-C compounds were adsorbed to phospholipid bilayers, the rate of drug release was too small to measure. The extraordinarily slow hydrolysis rates were even more evident with the membrane-bound p-nitrophenyl esters of our lipid-peptides. These hydrolysis rates were orders of magnitude slower than that of free phenylalanine p-nitrophenyl ester. It will be interesting to determine the number and structure of the amino acids required in the peptide spacer to achieve "normal" rates. These will depend upon the conformation of the peptide at the membrane surface and upon the minimum permissible enzymelmembrane contact distance. The work is now made possible by our synthetic route to lipidfpeptidel drug conjugates described herein. EXPERIMENTAL PROCEDURES

General Methods. Melting points were determined with a Thomas Hoover capillary melting point apparatus and are uncorrected. Fast atom bombardment (FAB)mass

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spectra were obtained on a VG 7 0 4 mass spectrometer using 3-nitrobenzyl alcohol (3-NBA) as the matrix. Elemental analyses were performed by Atlantic Microlab, Inc., P.O. Box 2288, Atlanta, GA. 'Hand 13CNMRspectra were recorded on a QE-300MHz spectrometer using CDC13 as the reference solvent unless otherwise indicated. Solvents were of reagent grade and dried over molecular sieves (4 A). THF was distilled under Nz from sodium benzophenone ketyl before use. All reactions were performed under N2 unless otherwise noted. N-Tridecanoylphenylalanine. To a solution of tridecanoic acid (4.29 g, 20 mmol) and triethylamine (2.8 mL, 20 mmol) in 50 mL of distilled THF at 0 OC under N2 was added slowly ethyl chloroformate (2.1 mL, 22 mmol). After the solution was stirred at 0 "C for 20 min, an ice-cold solution of sodium salt of phenylalanine, prepared from phenylalanine (3.3 g, 20 mmol) and sodium hydroxide (800mg, 20 mmol) in 40 mL of water, was added. The reaction mixture was warmed to room temperature, stirred for 19 h, and then acidified to pH -2 with 10% aqueous HCl(8). The mixture was poured into water (250 mL) and extracted with CHzClz (3 X 50 mL). The combined organic extracts were extracted again with brine (100 mL), dried over MgS04, filtered, and concentrated under reduced pressure. The crude residue was triturated with CH3CN and dried under vacuum to afford 4.82 g (67 %) of N-tridecanoylphenylalanine(mp 91-92 "C). 'H NMR (300 MHz, CDCl3): 6 0.88 (t, J = 6.6 Hz, 3H), 1.151.65 (m, 20 H), 2.18 (t, J = 7.5 Hz, 2 H), 3.10-3.20 (dd, J = 14.0 Hz, 6.3 Hz, lH), 3.20-3.30 (dd, J = 14.0 Hz, 5.5 Hz, lH), 4.90 (m, lH), 6.00 (d, J = 7.1 Hz, lH), 7.05-7.20 (m, 5H). 13C NMR (75.1 MHz, CDCl3): 6 14.1,22.7,25.5,29.1, 29.3, 29.4, 29.5, 29.6, 31.9, 36.4, 37.2, 53.1, 127.2, 128.6, 129.3, 135.6, 173.9, 174.9. LRMS (FAB): m/z 362 (M + HI+.Anal. Calcd for C22H35N03: C, 73.09; H, 9.76; N, 3.87. Found: C, 72.99; H, 9.70; N, 3.87. N-Tridecanoylglycylphenylalanine. To a solution of tridecanoic acid (4.29 g, 20 mmol) and triethylamine (2.8 mL, 20 mmol) in 45 mL of distilled THF at 0 "C under N2 was added slowly ethyl chloroformate (2.1 mL, 22 mmol). After the solution was stirred at 0 OC for 35 min, an ice-cold solution of sodium salt of glycylphenylalanine, prepared from glycylphenylalanine (4.4 g, 20 mmol) and sodium hydroxide (800 mg, 20 mmol) in 50 mL of water, was added. The reaction mixture was warmed to room temperature, stirred for 19 h, and then acidified to pH -2 with 10% aqueous HCl(8). The mixture was poured into water (300 mL) and extracted with CH2C12/CH30H (3 X 70 mL). The combined organic extracts were extracted again with brine (100 mL), dried over MgS04, filtered, and concentrated under reduced pressure. The crude residue was triturated with ether and dried under vacuum to afford 5.60 g (67% ) of N-tridecanoylglycylphenylalanine (mp 112-113 "C). lH NMR (300 MHz, CDC13): 6 0.88 (t, J = 6.6 Hz, 3H), 1.20-1.70 (m, 20 H), 2.22 (t, J = 7.5 Hz, 2 H), 2.95-3.25 (m, 2H), 3.60-4.00 (m, 2H), 7.10-7.30 (m, 5H). 13C NMR (75.1 MHz, CDCl3): 6 13.5,22.4,25.4,29.0, 29.1, 29.2, 29.4, 31.7, 35.7, 37.1, 42.3, 53.2, 126.6, 128.2, 129.1, 136.2, 169.6, 172.9, 175.1. LRMS (FAB): mlz 419 (M + HI+. Anal. Calcd for C24H38N20~0.5H20:C, 67.42; H, 9.19; N, 6.55. Found: C, 67.88; H, 9.03; N, 6.64. N-Tridecanoylglycylglycylphenylalanine.To a solution of tridecanoic acid (3.22 g, 15 mmol) and triethylamine (2.1 mL, 15 mmol) in 60 mL of distilled THF at 0 "C under Nz was added slowly ethyl chloroformate (1.6 mL, 16.5 mmol). After the solution was stirred at 0 OC for 150 min, an ice-cold solution of the sodium salt of glycylglycylphenylalanine, prepared from glycylglycyl-

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phenylalanine (4.19 g, 15 mmol) and sodium hydroxide (600 mg, 15 mmol) in 50 mL of water, was added. The reaction mixture was warmed to room temperature, stirred for 21 h, and then acidified to pH -2 with 10% aqueous HCl (8). The mixture was poured into water (150 mL) and extracted with CH2C12/CH30H (3 X 50 mL). The combined organic extracts were extracted again with brine (100 mL), dried over MgS04, filtered, and concentrated under reduced pressure. The crude residue was triturated with ether and dried under vacuum to afford 5.44 g (76 % ) of N-tridecanoylglycylglycylphenylalanine(mp 128-129 "C). 'H NMR (300 MHz, CDCl3): 6 0.88 (t, J = 6.6 Hz, 3H), 1.20-1.70 (m, 20 H), 2.27 (t, J = 7.5 Hz, 2H), 3.003.10(dd, J=14.0Hz,6.1Hz, lH),3.15-3.3O(dd,J=14.0 Hz, 5.0Hz, lH), 3.75-4.00 (m, 4H), 7.15-7.35 (m, 5H). 13C NMR (75.1 MHz, CDC13): 6 13.0, 21.9, 24.9, 28.6, 28.8, 28.9, 31.2, 35.2, 36.6, 41.5, 42.0, 53.1, 126.1, 127.7, 128.5, 136.1, 169.1, 170.0, 172.6, 175.0. LRMS (FAB): m/z 476 (M + HI+. Anal. Calcd for C ~ ~ H ~ ~ N ~ O V OC,. ~64.44; HZO: H, 8.74; N, 8.67. Found: C, 64.77; H, 8.76; N, 8.71. N-Heptadecanoylphenylalanine. To a solution of heptadecanoic acid (5.41 g, 20 mmol) and triethylamine (2.8 mL, 20 mmol) in 60 mL of distilled THF at 0 "C under N2 was added slowly ethyl chloroformate (2.1 mL, 22 mmol). After the solution was stirred at 0 "C for 20 min, an ice-cold solution of the sodium salt of phenylalanine, prepared from phenylalanine (3.3 g, 20mmol) and sodium hydroxide (800 mg, 20 mmol) in 50 mL of water, was added. The reaction mixture was warmed to room temperature, stirred for 19 h, and then acidified to pH -2 with 10% aqueous HCl(8). The mixture was poured into water (200 mL) and extracted with CHCl3 (3 X 100 mL). The combined organic extracts were extracted again with brine (200 mL), dried over MgS04, filtered, and concentrated under reduced pressure. The crude residue was triturated with C C 4and dried under vacuum to afford 5.85 g (70%) of N-heptadecanoylphenylalanine (mp 74-75 "C). 'H NMR (300 MHz, CDCl3): 6 0.88 (t, J = 6.5 Hz, 3H), 1.151.65 (m, 28 H), 2.18 (t, J = 7.5 Hz, 2H), 3.10-3.20 (dd, J = 14.0 Hz, 6.2 Hz, lH), 3.20-3.30 (dd, J =14.0 Hz, 5.5 Hz, lH), 4.90 (m, lH), 5.98 (d, J = 7.3 Hz, lH), 7.05-7.20 (m, 5H). '3C NMR (75.1 MHz, CDCl3): 6 14.1,22.7,25.6,29.1, 29.3, 29.4, 29.5, 29.7, 31.9, 36.4, 37.3, 53.1, 127.2, 128.6, 129.3, 135.6, 174.0, 174.7. LRMS (FAB): m/z 853 (M + 2Li - HI+.Anal. Calcd for Cz6H43N03-0.5H20: C, 73.20; H, 10.40; N, 3.28. Found: C, 73.77; H, 10.31; N, 3.33. N-Heptadecanoylglycylphenylalanine. To a solution of heptadecanoic acid (0.27 g, 1mmol) and triethylamine (0.14 mL, 1 mmol) in 6 mL of distilled THF at 0 "C under N2 was added slowly ethyl chloroformate (97 pL, 1mmol). After the solution was stirred at 0 "C for 20 min, an ice-cold solution of sodium salt of glycylphenylalanine, prepared from glycylphenylalanine (0.22 g, 1 mmol) and sodium hydroxide (0.04 mg, 1 mmol) in 6 mL water, was added. The reaction mixture was warmed to room temperature, stirred for 19 h, and then acidified to pH -2 with 10% aqueous HC1 (8). The mixture was poured into water (30 mL) and extracted with CHzC12/ CH30H (3 X 10mL), The combined organic extracts were extracted again with brine (30 mL), dried over MgS04, filtered, and concentrated under reduced pressure. The crude residue was triturated with ether and dried under vacuum to afford 0.30 g (64%) of N-heptadecanoylglycylphenylalanine (mp 117-118 "C). IH NMR (300 MHz, CDCl3): 6 0.88 (t,J = 6.6 Hz, 3H), 1.20-1.70 (m, 28 H), 2.22 (t, J = 7.5 Hz, 2H), 3.00-3.10 (dd, J = 14.0 Hz, 7.3Hz, lH), 3.15-3.25 (dd, J =14.0Hz, 5.2Hz, lH), 3.753.95 (m, 2H), 7.15-7.35 (m, 5H). 13C NMR (75.1 MHz,

Menger et al.

CDCl3): 6 14.2, 22.9, 25.8, 29.5, 29.6, 29.7, 29.9,32.1,36.3, 37.7,42.7,53.5,127.1,128.6,129.5,136.4,169.6,173.9,175.1. LRMS (FAB): m/z 476 (M + HI+. Anal. Calcd for C28H46N204: C, 70.85; H, 9.77; N, 5.90. Found: C, 70.63; H, 9.83; N, 5.85. N-Heptadecanoylglycylglycylphenylalanine.To a solution of heptadecanoic acid (3.23 g, 11.9 mmol) and triethylamine (1.70 mL, 11.9 mmol) in 100 mL of distilled THF at 0 "C under Nz was added slowly ethyl chloroformate (1.15 mL, 11.9 mmol). After the solution was stirred at 0 "C for 50 min, an ice-cold solution of sodium salt of glycylglycylphenylalanine, prepared from glycylglycylphenylalanine (3.34 g, 11.9 mmol) and sodium hydroxide (478 mg, 11.9 mmol) in 70 mL of water, was added. The reaction mixture was warmed to room temperature, stirred for 20 h, and then acidified to pH 2 with 10% aqueous HCl(8). The mixture was poured into water (200 mL) and extracted with CH&lz/CH30H (3 x 70 mL). The combined organic extracts were extracted again with brine (100 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The crude residue was triturated with ether and dried under vacuum to afford 5.35 g ( 8 2 % ) of N-heptadecanoylglycylglycylphenylalanine (mp 142-143 "C). 'H NMR (300 MHz, CDC13): 6 0.88 (t,J = 6.0 Hz, 3H), 1.20-1.70 (m, 28 H), 2.27 (t, J = 7.5 Hz, 2H), 3.00-3.10 (dd, J = 14.0 Hz, 6.1H~,1H),3.15-3.30(dd,J=14.0H~,5.0H~,1H),3.754.00 (m, 4H), 7.15-7.35 (m, 5H). 13C NMR (75.1 MHz, CDCl3): 6 14.2, 22.8, 25.7,29.5, 29.6, 29.8,32.0, 36.2, 37.4, 42.6,42.8,53.7,127.0,128.5,129.4,136.5,169.4,170.4,173.5, 175.2. LRMS (FAB): m/z 532 (M + HI+. Anal. Calcd for C30H49N305: C, 67.76; H, 9.29; N, 7.90. Found: C, 67.47; H, 9.31; N, 7.73. 2',3',5'-Tri-O-TBDMS-ara-C.A solution of ara-C (1.22 g, 5 mmol), TBDMSCl (4.56 g, 30 mmol), imidazole (2.04 g, 30 mmol), and DMAP (0.16 g, 1.25 mmol) in 40 mL of anhydrous CHzCl2 was stirred at room temperature for 4 days (7).The solvent was removed under reduced pressure, and the crude product was flash chromatographed on silica (20% CH30H/CHCl3) to afford 2.72 g of crude 2',3',5'tri-0-TBDMS-ara-C. It was used in the following experiment without further purification. M-(N-Tridecanoylphenylalany1)-ara-C.A solution of N-tridecanoylphenylalanine(564 mg, 1.56 mmol), tri0-TBDMS-ara-C (914 mg, 1.56 mmol), EEDQ (386 mg, 1.56 mmol), and pyridine (760 pL, 9.36 mmol) in 5 mL of anhydrous CHC13 was heated at 37 "C under nitrogen for 48 h (9). The mixture was cooled to room temperature and concentrated under reduced pressure. The oily crude product was flash chromatographed on a silica column (20 % CH30H/CHCl3). The resulting residue was treated, without further purification, with 1.0 M TBAF in THF (5.0 mL, 5.0 mmol) at room temperature for 2 h (IO).Flash chromatography on silica (20 % CH30H/CHC13) afforded 190 mg (217% ) of N4-(N-tridecanoylphenylalanyl)-ara-C (mp 183-184 "C). 'H NMR (300 MHz, CDCl3/CD30D, TMS = 0.0 ppm): 6 0.89 (t,J = 6.6 Hz, 3H), 1.15-1.60 (m, 20 H), 2.18 (t, J = 7.5 Hz, 2H), 2.90-3.30 (m, 2H), 3.753.95 (m, 2H), 4.05-4.35 (m, 3H), 6.23 (d, J = 5.0 Hz, lH), 7.15-7.35 (m, 5H), 7.45 (d, J = 7.4 Hz, lH), 8.27 (d, J = 7.4 Hz, 1H). 13CNMR (75.1 MHz, CDCl3/CD30D, CDC13 = 77 ppm): 6 13.0, 21.9, 25.0, 28.4, 28.6, 28.7, 28.9, 31.2, 35.2,36.7,54.7,54.8,60.9,74.4,76.3,85.4,87.7,95.5,126.2, 127.7,128.4,135.6,146.1,155.7,161.8,171.8,174.3. LRMS (FAB): mlz 593 ( M + L i ) + . Anal. Calcd for C31H46N40yH20: C, 61.57; H, 8.00; N, 9.26. Found: C, 61.64; H, 7.75; N, 9.09. M-(N-Tridecanoylglycylphenylalany1)-ara-C.A

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Technical Notes

solution of N-tridecanoylglycylphenylalanine(628mg, 1.5 mmol), tri-0-TBDMS-ara-C (879 mg, 1.5 mmol), EEDQ (371 mg, 1.5 mmol), and pyridine (728 pL, 9.0 mmol) in 10 mL anhydrous CHCl3 was heated at 50 "C under nitrogen for 48 h (9). The mixture was cooled to room temperature and concentrated under reduced pressure. The oily crude product was flash chromatographed on a silica column (20% CH30H/CHC13). The resulting residue was treated, without further purification, with 1.0M TBAF in THF (10.0 mL, 10.0 mmol) at room temperature for 2 h (10). Flash chromatography on silica (20% CH30H/ CHC13) afforded 220 mg (23%) of N4-(N-tridecanoylglycylphenylalany1)-ara-C(mp 120-121 "C). 'H NMR (300 MHz, CDC13/CD30D, TMS = 0.0 ppm): 6 0.88 (t, J = 6.6 Hz, 3H), 1.20-1.70 (m, 20 H), 2.22 (t, J = 7.5 Hz, 2H), 2.95-3.25 (m, 2H), 3.75-4.35 (m, 7H), 4.80-5.00 (m, lH), 6.23 (d, J = 5.0 Hz, lH), 7.15-7.45 (m, 6H), 8.27 (d, J = 7.4Hz), 1H). 13CNMR (75.1MHz, CDCldCD30D, CDC13 = 77 ppm): 6 13.4, 22.2, 25.2, 28.8, 28.9, 29.0, 29.1, 31.4, 35.6,37.2,42.1,55.0,61.1,74.6,85.5,87.9,95.8,126.6,128.1, 128.7,135.4,146.3,155.8,161.9,169.6,171.5,174.8. LRMS (FAB): m l z 644 ( M + H ) + . Anal. Calcd for C33H49N50g0.5H20: C, 60.72; H, 7.72; N, 10.73. Found: C, 60.96; H, 7.69; N, 10.79. N4- ( N - T r i d ec a n o y 1 g1y c y 1g 1y c y 1p hen y 1 a 1an y1)-ara-C. A solution of N-tridecanoylglycylglycylphenylalanine (950 mg, 2.0 mmol), tri-0-TBDMS-ara-C (1.17g, 2.0mmol),EEDQ (494mg, 2.0mmol),andpyridine (970 pL, 12.0 mmol) in 10 mL of anhydrous CHC13 was heated at 50 "C under nitrogen for 51 h (9). The mixture was cooled to room temperature and concentrated under reduced pressure. The oily crude product was flash chromatographed on a silica column (20% CH30H/ CHC13). The resulting residue was treated, without further purification, with 1.0 M TBAF in THF (15.0 mL, 15.0 mmol) at room temperature for 2 h (10). Flash chromatography on silica gel (20% CH3OH/CHC13)afforded 435 mg (31% ) of N4-(N-tride~an~ylgly~ylglycylphenylalanyl)ara-C (mp 122-123 "C). lH NMR (300 MHz, CDC13/CD3OD,TMS=O.Oppm): 60.88(t, J=6.6Hz,3H),1.15-1.75 (m, 20 H), 2.27 (t, J = 7.5 Hz, 2H), 3.00-3.30 (m, 2H), 3.75-4.35 (m, 7H), 4.75-4.95 (m, lH), 6.23 (d, J = 5.0 Hz, lH), 7.15-7.30 (m, 5H), 7.40 (d, J = 7.4 Hz, lH), 8.27 (d, J = 7.4 Hz, 1H). 13C NMR (75.1 MHz, CDC13/CD30D, CDCl3 = 77 ppm): 6 13.3,14.3, 22.1, 25.1,28.8,28.9,29.0, 29.1,31.4,35.4,36.7,42.0,42.4,55.4,61.0,65.4,74.6,76.3, 85.4,87.8,95.7,126.4,128.0,135.8,146.4,155.7,161.8,169.8,

170.6, 171.5, 175.2. LRMS (FAB): m/z 707 (M + Li)+. Anal. Calcd for C35H52N609"20: C, 58.48; H, 7.57; N, 11.69. Found: C, 58.34; H, 7.64; N, 11.61. N4-(N-Heptadecanoylphenylalany1)-ara-C.A solution of N-heptadecanoylphenylalanine (508 mg, 1.21 mmol), tri-0-TBDMS-ara-C (713 mg, 1.21 mmol), EEDQ (301 mg, 1.21 mmol), and pyridine (590 pL, 7.3 mmol) in 5 mL of anhydrous CHC13 was heated at 37 "C under nitrogen for 48 h (9). The mixture was cooled to room temperature and concentrated under reduced pressure. The oily crude product was flash chromatographed on a silica column (20% CH30H/CHCl3). The resulting residue was treated, without further purification, with 1.0M TBAF in THF (7.0 mL, 7.0 mmol) at room temperature for 2 h (10). Flash chromatography on silica (20% CH3OH/ CHC13) afforded 368 mg ( 4 7 % ) of N4-(N-heptadecanoylphenylalany1)-ara-C(mp 161-162 "C). lH NMR (300 MHz, CDC13/CD3OD, TMS = 0.0 ppm): 6 0.89 (t-like, 3H), 1.10-1.60 (m, 28 H), 2.18 (t-like, 2H), 2.803.30 (m, 2H), 3.70-4.40 (m, 5H), 4.60-5.00 (m, lH), 6.23 (s, lH), 7.00-7.50 (m, 6H), 8.24 (d, J = 7.4 Hz, 1H). 13C

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NMR (75.1 MHz, CDCL/CD30D, CDCL = 77 ppm): 6 13.0,21.9,25.0,28.4,28.6,28.7,31.2,35.1,36.7,54.7,54.8, 60.9, 74.4, 76.3,85.4, 87.6,95.4, 126.2, 127.7, 128.4, 135.6, 146.1,148.0,155.7,161.8,171.8,174.3. LRMS (FAB): m/z 649 (M + Li)+. Anal. Calcd for C35H54N40~2H20:C, 64.49; H, 8.50; N, 8.60. Found: C, 64.61; H, 8.41; N, 8.38. N-(N-Heptadecanoylglycylphenylalany1)-ara-C.A solution of N-heptadecanoylglycylphenylalanine(712 mg, 1.5 mmol),tri-0-TBDMS-ara-C (879mg, 1.5mmol),EEDQ (371 mg, 1.5 mmol), and pyridine (730 pL, 9.0 mmol) in 10 mL of anhydrous CHCl3 was heated at 50 "C under nitrogen for 48 h (9). The mixture was cooled to room temperature and concentrated under reduced pressure. The oily crude product was flash chromatographed on a silica column (20 % CH30H/CHC13). The resulting residue was treated, without further purification, with 1.0 M TBAF in THF (10.0 mL, 10.0 mmol) at room temperature for 2 h (10). Flash chromatography on silica (20% CH30H/ CHCl3) afforded 252 mg (24%)of N4-(N-heptadecanoylglycylphenylalany1)-ara-C(mp 135-136 "C). lH NMR (300 MHz, CDCl$CD3OD, TMS = 0.0 ppm): 6 0.88 (t, J = 6.6 Hz, 3H), 1.20-1.70 (m, 28 H), 2.22 (t, J = 7.5 Hz, 2H), 7.10-7.35 (m, 5H), 7.42 (d, J = 7.4 Hz, lH), 8.27 (d, J = 7.4 Hz, 1H). 13CNMR (75.1 MHz, CDCl3/CD3OD,CDC13 = 77 ppm): 6 13.6, 22.3, 25.3, 29.0, 29.1, 29.2, 29.3, 31.6, 35.7,37.2,42.2,55.1,61.2,74.7,85.8,88.0,95.9,126.7,128.2, 128.9,135.5,146.4,162.0,162.9,169.9,171.5,174.7. LRMS (FAB): m / z 706 ( M + L i ) + . Anal. Calcd for C37H57N50gH20: C, 61.90; H, 8.28; N, 9.76. Found: C, 61.94; H, 8.17; N, 9.63. N4-(N-Heptadecanoylglycylglycylphenylalany1)ara-C. A solution of N-heptadecanoylglycylglycylphenylalanine (1.14g, 1.5mmol),tri-0-TBDMS-ara-C (880 mg, 1.5 mmol), EEDQ (372 mg, 1.5 mmol), and pyridine (730 pL, 9.0 mmol) in 1 2 mL of anhydrous CHC13 was heated at 50 "C under nitrogen for 72 h (9). The mixture was cooled to room temperature and concentrated under reduced pressure. The oily crude product was flash chromatographed on a silica column (20% CH30H/ CHCl3). The resulting residue was treated, without further purification, with 1.0 M TBAF in THF (14.0 mL, 14.0 mmol) at room temperature for 2 h (10). Flash chromatography on silica (20% CH30H/CHC13) afforded 360 mg (32 % ) of N4-(N-heptadecan~ylglycylglycylphenylalanyl)ara-C (mp 118-119 "C). lH NMR (300 MHz, CDCls/CD3OD,TMS=O.Oppm): 60.88(t, J=6.6Hz,3H),1.15-1.70 (m, 28 H), 2.27 (t, J = 7.5 Hz, 2H), 3.00-3.30 (m, 2H), 3.75-4.35 (m, 7H), 4.75-4.95 (m, lH), 6.23 (d, J = 5.0 Hz, lH), 7.15-7.35 (m, 5H), 7.42 (d, J = 7.4 Hz, lH), 8.27 (d, J = 7.4 Hz, 1H). 13C NMR (75.1 MHz, CDC13/CD30D7 CDCl3 = 77 ppm): 6 13.3,14.3, 22.1, 25.1,28.8,28.9,29.0, 29.2,35.4,36.7,42.0,42.4,55.3,61.0,65.4,74.6,76.4,85.5, 87.8, 95.7, 126.4, 128.0, 128.7, 135.7, 146.4, 155.7, 161.8, 170.0, 170.5, 171.5, 175.1. LRMS (FAB): m/z 764 (M Li)+. Anal. Calcd for C~~&&O$I-~H~O: C, 59.07; H, 8.14; N, 10.60. Found: C, 58.71; H, 7.86; N, 10.47.

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ACKNOWLEDGMENT

This work was supported by the National Institutes of Health (GM 21457). LITERATURE CITED (1) Bodey, G. P., Freireich, E. J., Monto, R. W., and Hewlett, J. S. (1969)Cytosine Arabinoside (NSC-63878)Therapy for Acute Leukemia in Adults. Cancer Chemother.Rep. (Part I ) 53,59. ( 2 ) Holland, J. F., and Glidewell, 0. (1970) Complementary

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